Analysis of Saccharides
Technical Note
The major components of food include carbohydrates, lipids, proteins, vitamins
and inorganic compounds. Carbohydrates are an important energy resource. The
basic structure is made of long chains of saccharides. By using different separation
modes, various types of saccharides can be separated by high-performance liquid
chromatography (HPLC). Recommended HPLC columns for saccharide types are
listed in this table.
Classification 1
Classification 2
Example
Monosaccharides・Disaccharides
Saccharide derivatives With dissociating group
Recommended HPLC columns
Glucose
Phosphorylated
saccharide
Without dissociating group
Sugar alcohol
Oligosaccharides
Trehalose
Polysaccharides
Hyaluronan
Hydrolysate of polysaccharide
Enzyme ecomposer
Molecular weight under 2000 Catechins
Glycoside
Molecular weight above 2000 Glycoprotein
Sugar-D
HILIC (Triazole)
Sugar-D
Sugar-D
Diol-II series / CNT series
C18-MS-II etc. C18 series Cholester
C18-MS-II etc. C18 series Cholester
Column for Protein separation such as Protein-R
Analysis of mono and oligosaccharides
Sugar-D is designed for separation of saccharides. It is the most suitable HPLC column for the analysis of mono- and
oligosaccharides. Its quantitative capability and durability are excellent when comparing with conventional columns.
Component of soft drink
Component of chocolate
COSMOSIL Application Data
COSMOSIL Application Data
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Sugar-D
4.6mmI.D.-250mm
Acetonitrile / H2O = 75/25
1.0 ml/min
30°C
RI
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Sugar-D
4.6mmI.D.-250mm
Acetonitrile / H2O = 75/25
1.0 ml/min
30°C
RI
Sample:
1; Fructose
2; Glucose
3; Sucrose
4; Maltose
Sample:
1; Sucrose
2; Lactose
(10㱘g)
(10㱘g)
(10㱘g)
(10㱘g)
(10㱘g)
(10㱘g)
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NACALAI TESQUE, INC
AP-0326
Component of GUM
AP-0324
Component of sports drink
COSMOSIL Application Data
COSMOSIL Application Data
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Sugar-D
4.6mmI.D.-250mm
Acetonitrile / H2O = 75/25
1.0 ml/min
30°C
RI
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Sample:
1; meso-Erythritol
2; Xylitol
3; Palatinose
4; Maltitol
Sample:
1; meso-Erythritol
2; Fructose
3; Glucitol
4; Glucose
5; Sucrose
6; Maltose
7; Maltotriitol
8; Maltotriose
(10㱘g)
(10㱘g)
(10㱘g)
(10㱘g)
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AP-0323
Sugar-D
(4.6mmI.D.-250mm) 㬍2
Acetonitrile / H2O = 75/25
1.0 ml/min
50°C
RI
(10㱘g)
(10㱘g)
(10㱘g)
(10㱘g)
(10㱘g)
(10㱘g)
(10㱘g)
(10㱘g)
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AP-0327
Analysis of Saccharide Derivatives
There are two kinds of Saccharide derivatives. One has a dissociating group, such as Phosphorylated saccharide. The other
one has none, such as sugar alcohol.
(1) Saccharide derivatives with dissociating group (Phosphorylated saccharide etc.)
Saccharide derivatives with dissociating group can be well separated by COSMOSIL HILIC with high reproducibility. Sugar-D
can be used but certain buffer may cause low reproducibility.
Phosphorylated saccharide
D-Glucuronic Acid
COSMOSIL Application Data
COSMOSIL Chromatogram Index
Column:
HILIC
Column size: 4.6mmI.D.-250mm
Mobile phase: Acetonitrile/ 20mmol/l Phosphate
buffer(pH7.0) = 60/40
Flow rate:
1.0 ml/min
Temperature: 30°C
Detection:
RI
Sample:
CAS No.:
Molecular formula:
Column:
Column size:
Mobile phase:
Sample:
1; D㵥Fructose㵥6㵥phosphate (50㱘g)
2; D-Glucose-6-phosphate (50㱘g)
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Flow rate:
Temperature:
Detection:
Attenuation:
Sample conc.:
Injection volume:
Retention time:
Capacity factor:
D-Glucuronic Acid
HO
O
[6556-12-3]
C6H10O7
OH
HILIC
OH
4.6mmI.D.-250mm
Acetonitrile/ 10mmol/l
Phosphate buffer(pH7)=50/50
1.0 ml/min
30°C
UV210 nm
0.128 aufs
10.0mg/ml
1.0µl
5.45min
0.92
O
OH
OH
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AP-0317
(2) Saccharide derivatives without dissociating group (Sugar alcohol etc.)
Saccharide derivatives without dissociating group can be separated by Sugar-D.
Component of GUM
N-Acetyl-D-glucosamine
COSMOSIL Application Data
COSMOSIL Chromatogram Index
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Sample:
CAS No.:
Molecular formula:
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Attenuation:
Sample conc.:
Injection volume:
Retention time:
Capacity factor:
Sugar-D
4.6mmI.D.-250mm
Acetonitrile / H2O = 75/25
1.0 ml/min
30°C
RI
Sample:
1; Xylitol
2; Mannitol
3; Maltitol
4; Palatinit
5; Maltotriitol
6; Maltotertaitol
(10㱘g)
(10㱘g)
(10㱘g)
(10㱘g)
(10㱘g)
(10㱘g)
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AP-0325
N-Acetyl-D-glucosamine
[7512-17-6]
C8H15NO6
Sugar-D
4.6mmI.D.-250mm
Acetonitrile/ H2O=80/20
1.0 ml/min
30°C
ELSD
Gain=6,Atten=9
1.0mg/ml
3.0µl
6.98min
1.64
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Analysis of Oligosaccharides
Oligosaccharides benefit human health in various ways, such as increasing beneficial intestinal bacteria…etc. Therefore, they
are widely used in health food. Decasaccharide analysis can be conducted by COSMOSIL Sugar-D.
Cyclodextrin
Fructooligosaccharides
COSMOSIL Application Data
COSMOSIL Application Data
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Sugar-D
4.6mmI.D.-250mm
Acetonitrile / H2O = 65/35
1.0 ml/min
30°C
RI
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Sugar-D
4.6mmI.D.-250mm
Acetonitrile / H2O = 70/30
1.0 ml/min
30°C
RI
Sample:
1; 㱍-Cyclodextrin
2; 㱎-Cyclodextrin
3; 㱏-Cyclodextrin
Sample:
Fructooligosaccharides (50㱘g)
1; 1-Kestose
2; Nystose
3; 1-Fructofuranosyl-D-nystose
(10㱘g)
(10㱘g)
(10㱘g)
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AP-0320
AP-0336
Isomaltooligosaccharides
Lactulose
COSMOSIL Application Data
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Sugar-D
4.6mmI.D.-250mm
Acetonitrile / H2O = 75/25
1.0 ml/min
30°C
RI
Sample:
Isomaltooligosaccharides (50㱘g)
(Isomaltose, Isomaltotriose, Panose etc.)
COSMOSIL Chromatogram Index
Sample:
CAS No.:
Molecular formula:
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Attenuation:
Sample conc.:
Injection volume:
Retention time:
Capacity factor:
Lactulose
[4618-18-2]
C12H22O11
Sugar-D
4.6mmI.D.-250mm
Acetonitrile/ H2O=80/20
1.0 ml/min
30°C
ELSD
Gain=6,Atten=9
1.0mg/ml
5.0µl
13.96min
4.31
HO
HO
H
HO
HO
H
HO
H
H
OH
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AP-0322
Saponin
COSMOSIL Application Data
COSMOSIL Application Data
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Sugar-D
4.6mmI.D.-250mm
Acetonitrile / H2O = 65/35
1.0 ml/min
30°C
RI
Sample:
1; Glucose
2; Maltose
3; Maltotriose
4; Maltotetraose
5; Maltopentaose
6; Maltohexaose
7; Maltoheptaose
H
O
H
OH
OH
O
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Maltooligosaccharide
O
H
H
Sugar-D
4.6mmI.D.-150mm
Acetonitrile/ H2O = 75/25
1.0 ml/min
30°C
UV210nm
Sample:
(10㱘g)
(10㱘g)
(10㱘g)
(10㱘g)
(10㱘g)
(10㱘g)
(10㱘g)
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AP-0331
㩍㩨㨺㩊ឭଏ
ർ㒽ᄢቇ⮎ቇㇱ Ꮉ↰ᐘ㓶వ↢
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AP-0451
D-Trehalose
Xylooligosaccharides
COSMOSIL Application Data
COSMOSIL Chromatogram Index
D-(+)-Trehalose
[6138-23-4]
C12H22O11
Sugar-D
4.6mmI.D.-250mm
Acetonitrile/ H2O=80/20
1.0 ml/min
30°C
ELSD
Gain=6,Atten=9
1.0mg/ml
5.0µl
18.26min
5.95
Sample:
CAS No.:
Molecular formula:
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Attenuation:
Sample conc.:
Injection volume:
Retention time:
Capacity factor:
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Sugar-D
4.6mmI.D.-250mm
Acetonitrile / H2O = 75/25
1.0 ml/min
30°C
RI
Sample:
Xylooligosaccharides (50㱘g)
(Xylobiose, Xylotriose etc.)
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AP-0321
Size Exclusion Analysis of Polysaccharide
Polysaccharides such as hyaluronan and chondroitin are widely used in food, cosmetics and medicine.
COSMOSIL Diol-II is excellent for the analysis of polysaccharides in gel filtration mode. COSMOSIL CNT column is suitable for
large molecular weight compounds (approximately one million) such as hyaluronan.
Chondroitin Sulfate A
Chondroitin Sulfate C
COSMOSIL Chromatogram Index
COSMOSIL Chromatogram Index
Sample:
CAS No.:
Column:
Column size:
Mobile phase:
Sample:
CAS No.:
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Attenuation:
Sample conc.:
Injection volume:
Retention time:
Chondroitin Sulfate A
[39455-18-0]
5Diol-300-II
7.5mmI.D.-300mm
20mmol/l Phosphate buffer,
100mmol/l Na2SO4(pH6.7)
1.0 ml/min
30°C
RI
4x10-5 RIU/FS
20.0mg/ml
1.0µl
6.45min
Flow rate:
Temperature:
Detection:
Attenuation:
Sample conc.:
Injection volume:
Retention time:
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Hyaluronic Acid
COSMOSIL Application Data
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Sample:
CNT-1000
7.5mmI.D.-300mm
20mmol/l Phosphate buffer(pH7),100mmol/l Na2SO4
1.0 ml/min
30°C
RI
Hyaluronic Acid
(100㱘g)
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AP-1081
Chondroitin Sulfate C
[12678-07-8]
5Diol-300-II
7.5mmI.D.-300mm
20mmol/l Phosphate buffer,
100mmol/l Na2SO4(pH6.7)
1.0 ml/min
30°C
RI
4x10-5 RIU/FS
20.0mg/ml
1.0µl
6.14min
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Analysis of Polysaccharide Hydrolysate
(1) Chondroitin
Although polysaccharides can be analyzed by gel filtration as described in 4, detection of hydrolysates is a commonly used
enzymatic digestion technique.
COOH
O
OH
O
OR2
OH
OR1
O
O
Chondroitinase AC-II
HN
Chondroitin
O
COOH
O
OH
O
OR2
OR1
(Major hydrolysate)
(R1: H, R2: SO3-)
(R1: SO3-, R2: H)
ΔDi-4S
OH
n
OH
O
HN
Δ-Disaccharide
O
ΔDi-6S
Enzyme digest of Chondroitin Sulfate A
Enzyme digest of Chondroitin Sulfate C
COSMOSIL Application Data
COSMOSIL Application Data
Column:
Cholester
Column size: 4.6mmI.D.-150mm
Mobile phase: A; 1mmol/l Tetrabutylammonium bisulfate/H2O
B; 1mmol/l Tetrabutylammonium bisulfate
-Acetonitrile/H2O=67/33
B conc. 20%㸢65%(7min)㸢65%(12min)
Flow rate:
1.0 ml/min
Temperature: 40°C
Detection:
UV240nm
Column:
Cholester
Column size: 4.6mmI.D.-150mm
Mobile phase: A; 1mmol/l Tetrabutylammonium bisulfate/H2O
B; 1mmol/l Tetrabutylammonium bisulfate
-Acetonitrile/H2O=67/33
B conc. 20%㸢65%(7min)㸢65%(12min)
Flow rate:
1.0 ml/min
Temperature: 40°C
Detection:
UV240nm
Sample:
Sample:
Chondroitin Sulfate A
Chodrotinase AC-II digested
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AP-1082
Chondroitin Sulfate C
Chodrotinase AC-II digested
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AP-1083
(2) Derivatization of Sugar chain
Generally, Sugar chains have low ultraviolet absorption so that highly sensitive detection is difficult to achieve. In such cases,
highly sensitive detection can be achieved by PA derivatization and fluorescence.
PA-Glucose Oligomer
COSMOSIL Application Data
Column:
5C18-PAQ
Column size: 4.6mmI.D.-150mm
Mobile phase: A: 20mmol/l Acetate buffer(pH3.3)
B: 20mmol/l Acetate buffer(pH3.3)+0.5%Butanol
B(0㸢100%) 45min Linear gradient
Flow rate:
1.0 ml/min
Temperature: 30°C
Detection:
FLS at Ex.320nm
Em. 400nm
Sample:
PA-Glucose Oligomer(DP=3-22)
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AP-0379
Analysis of Glycoside
(1) Molecular weight under 2000
Most of glycosides, such as saponin and cardiac glycosides, are physiologically active substance. These substances are
hydrophobic so they can be separated by C18 reversed phase columns.
Isoflavone glucoside
D- Amygdalin
COSMOSIL Application Data
COSMOSIL Application Data
Column:
5C18-MS-II
Column size: 4.6mmI.D.-150mm
Mobile phase: A: Acetonitrile/ 20mmol/l Phosphate
buffer(pH2.5) = 10/90
B: Acetonitrile/ 20mmol/l Phosphate
buffer(pH2.5) = 50/50
B conc. 0㸢100% 20min Linear gradient
Flow rate:
1.0 ml/min
Temperature: 30°C
Detection:
UV260nm
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Sample:
1; Daidzin
2; Glycitin
3; Genistin
4; Daidzein
5; Glycitein
6; Genistein
(0.05㱘g)
(0.05㱘g)
(0.05㱘g)
(0.05㱘g)
(0.075㱘g)
(0.025㱘g)
Sample:
5C18-MS-II
4.6mmI.D.-150mm
Methanol/ 50mmol/l NaH2PO4 = 1/5
0.8 ml/min
45°C
UV210nm
1; D㵥(-)-Amygdalin
(2.0㱘g)
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AP-0275
Ginsenosides
AP-0944
Ginsenosides
COSMOSIL Application Data
COSMOSIL Application Data
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Sample:
5C18-MS-II
4.6mmI.D.-150mm
Acetonitrile/ H2O = 30/70
1.0 ml/min
40°C
UV203nm
1; Ginsenoside Rb1
2; Ginsenoside Rc
Sample:
(2.5㱘g)
(2.5㱘g)
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AP-0923
Cardiac glycosides
COSMOSIL Application Data
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Sample:
5C18-AR-II
4.6mmI.D.-150mm
Methanol/ H2O = 70/30
1.0 ml/min
30°C
UV230nm, 0.32AUFS
1; Digitoxigenin
2; Digitoxin
(2.5㱘g)
(5.0㱘g)
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AP-0083
5C18-MS-II
4.6mmI.D.-150mm
Acetonitrile/ H2O = 20/80
1.0 ml/min
30°C
UV203nm
1; Ginsenoside Rg1
2; Ginsenoside Re
(2.5㱘g)
(2.5㱘g)
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AP-0920
Some analytes can not be separated adequately by C18, but they can be well separated using COSMOSIL Cholester column.
Saikosaponins
COSMOSIL Application Data
5C18-MS-II
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Sample:
Cholester
4.6mmI.D.-150mm
Acetonitrile/ H2O = 45/55
1.0 ml/min
30°C
ELSD, Gain=6
1; Saikosaponin c
2; Saikosaponin a
3; Saikosaponin b2
4; Saikosaponin b1
5; Saikosaponin d
(1.5㱘g)
(1.5㱘g)
(1.5㱘g)
(1.5㱘g)
(1.5㱘g)
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AP-1021
Catechin
COSMOSIL Application Data
5C18-MS-II
Column:
Column size: 4.6mmI.D.-150mm
Mobile phase: A: Acetonitrile/ 20mmol/l Phosphate buffer(pH2.5) = 10/90
B: Acetonitrile/ 20mmol/l Phosphate buffer(pH2.5) = 30/70
B conc. 0㸢100% 20min Linear gradient
Flow rate:
1.0 ml/min
Temperature: 30°C
Detection:
UV280nm
Sample:
1; Gallocatechin(GC)
(0.80mg/ml)
2; Caffeine
(0.08mg/ml)
3; Epigallocatechin(EGC)
(0.80mg/ml)
4; Catechin (C)
(0.40mg/ml)
5; Epicatechin(EC)
(0.40mg/ml)
6; Epigallocatechin gallate(EGCG)
(0.20mg/ml)
7; Gallocatechin gallate(GCG)
(0.40mg/ml)
8; Epicatechin gallate(ECG)
(0.20mg/ml)
9; Catechin gallate(CG)
(0.20mg/ml)
Injection Vol. 1.0㱘l
Cholester
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AP-1072
(2) Molecular weight above 2000
Glycoproteins with high molecular weight can be separated by using Protein-R. Application data of avidin analysis by using
Protein-R is shown below.
Avidin
COSMOSIL Chromatogram Index
Sample:
Source:
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Attenuation:
Sample conc.:
Injection volume:
Retention time:
Avidin
Egg White
Protein-R
4.6mmI.D.-150mm
A:0.05%TFA-H2O
B:0.05%TFA-60%Acetonitrile
B conc. 0㸢100% 20min Linear gradient
1.0 ml/min
30°C
UV 220nm
0.256 aufs
2.0mg/ml
2.5µl
16.22min
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<Reference> Analysis of food additives
Food additives are substances added to food to preserve flavor or enhance its taste and appearance. Saccharides are usually
used in food additives
The ingredient standards of food additives are stated by “Specifications and Standards for Food additives”. It indicated
extensive amounts of analysis methods by using Gas Chromatography. However, many HPLC methods also apply to refractory
substances and high temperature analysis condition required substances. Below is an analysis method for Glycyrrhizic Acid
described in the “Specifications and Standards for Food additives”.
Glycyrrhizic Acid
COSMOSIL Application Data
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Sample:
<< Analysis method for crude and purified licorice >>
5C18-MS-II
4.6mmI.D.-250mm
Acetonitrile/ 2%H3PO4 = 40/60
1.0 ml/min
30°C
UV254nm
1; Glycyrrhizic Acid
2; Propyl p-Hydroxybenzoate
(Described in “Specifications and Standards for Food additives”)
(5.0㱘g)
(1.0㱘g)
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AP-0926
Detection:
UV254nm
Column:
Packed with octadecyl bonded silica gel for 5 ~ 10 μm LC.
Column Size: 4 ~ 6mmI.D.- 150 ~ 300 mm
Temperature:
40oC
Mobile Phase:
Acetic acid (1→50) / acetonitrile = 3: 2
Flow rate:
Adjust the retention time of glycyrrhizic acid to 10min.
Selection of Column: Dissolve reference standard of Glycyrrhizic Acid (5 mg)
and Propyl p-Hydroxybenzoate (1 mg) in 20ml of 50% ethanol. A column which
can completely separate Glycyrrhizic Acid and Propyl p-Hydroxybenzoate when
inject 20 ul of the above solvent could be used.
Price list
Product Name
Separation mode
2.0-150
Product No.
2.0-250
Product No.
Sugar-D
HILIC
Hydrophilic
05689-31
Diol-120-II
Diol-300-II
C18-MS-II
Gel filtration
Cholester
Protein-R
Reversed phase
07054-71
38025-91
05971-11
07489-91
05761-61
05972-01
3.0-150
Product No.
05690-91
07871-61
34245-31
05973-91
3.0-250
Product No.
05691-81
07872-51
34254-11
05974-81
06514-71
4.6-150
Product No.
05395-71
07056-51
38019-81
05976-61
06526-21
4.6-250
Product No.
05397-51
07057-41
38020-41
05977-51
06527-11
7.5-300
Product No.
7.5-600
Product No.
10-250
Product No.
05692-71
07059-21
38023-11
05979-31
06530-51
20-250
Product No.
05693-61
07060-81
38024-01
05982-71
06532-31
38050-51
38053-21
38051-41
38054-11
Other size may be available. Please enquire.
For research use only, not intended for diagnostic or drug use.
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Nijo Karasuma, Nakagyo-ku, Kyoto 604-0855 JAPAN
: +81-(0)75-251-1730
TEL
: +81-(0)75-251-1763
FAX
Website : www.nacalai.com
E-mail : [email protected]
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